1. Field of the Invention
This invention relates to assay methods, and reagent means for use therein, of the homogeneous and heterogeneous specific binding type for determining qualitatively or quantitatively a ligand in a liquid medium. In particular, the invention relates to an improved nonradioisotopic binding assay employing a novel enzyme substrate label.
2. Description of the Prior Art
In German Offenlegungschriften Nos. 2,618,419 and 2,618,511, corresponding respectively to U.S. patent applications Ser. Nos. 667,982 now abandoned and 667,996, both filed Mar. 18, 1976 and assigned to the present assignee, there are described homogeneous and heterogeneous specific binding assays employing an enzyme-cleavable substrate label. In exemplified embodiments there are disclosed the use of fluorogenic-labeled conjugates comprising umbelliferone or fluorescein coupled via an ester group to a ligand under assay or to a binding partner therefor. The amount of labeled conjugate in the bound-species and/or free-species resulting from the binding reaction system employed is determined by addition of an esterase which cleaves the ester group linking the umbelliferone or fluorescein residue to the ligand or binding partner to release the free fluorescent products, umbelliferone and fluorescein, respectively. The rate of fluorescence production, which follows the rate of release of the fluorescent product, is a function of the amount of ligand in the liquid medium tested.
Performance of this assay depends upon the ability to determine the amount of labeled conjugate which results in either the bound-species or the free-species relative to the amount initially introduced. Where the measured character of the labeled conjugate in the bound-species is essentially indistinguishable from that in the free-species, the two species must be physically separated in order to complete the assay. This type of binding assay follows what is conventionally known as a "heterogeneous" format. On the other hand, where the measured character of the labeled conjugate in the two species is distinguishable, a "homogeneous" format can be followed if desired and the separation step avoided.
While the above described binding assays employing an enzyme-cleavable substrate label offer a generic, novel approach to the pertinent art, the application of the assays to the detection of ligands in certain types of liquid media using the ester linked labeled conjugate is restricted. For example, the ester based assay has been found to be inconvenient for the detection of ligands appearing in the milligram per liter concentration range in physiological fluids such as serum and plasma. It has been found in this situation that the fluid under assay can contain a high endogenous esterase activity and, independently, the ester linked conjugate can exhibit a significant instability as the result of background hydrolysis under the conditions of the assay, which are usually alkaline.